a, Image analysis pipeline to detect F-actin bundles. The core of the pipeline is a steerable filter that enhances the contrast of curvilinear image features. White squares outline areas shown at a higher magnification. Scale bars, 20 μm (main panel), 5 μm (magnification). b–d, F-actin organization of HBEC76 cells, and bundle detection after plating cells on normal-adhesive and low-adhesive substrates (b), on stiff and soft substrates (c) or after latrunculin A (200 nM) treatment (d). Positions of magnified regions are indicated by red boxes. Scale bars, 10 μm. Representative images from a single imaging experiment. e, F-actin organization of untransformed HBECs versus NSCLC cells. Right panels show filament detection. Scale bar, 10 μm. Representative images from a single imaging experiment. f, g, Quantification of F-actin bundle length (f) or intensity (g) in HBECs (HBEC30, n = 21 cells; HBEC34, n = 15 cells; and HBEC76, n = 16 cells) versus NSCLC cells (HCC4087, n = 11 cells; H2009, n = 22 cells; and H1819, n = 11 cells). h, Effect of latrunculin A treatment on oxidative phosphorylation of HBEC76 (black), HCC4087 (red) and HCC827 (orange) cells. Mean oxidative phosphorylation rates normalized to control ± s.d. are shown for each group. Data are from three independent experiments. Data in f, g are shown as a box (median ± 25–75%) and whisker (maximum to minimum values) plot, and statistical significance was assessed using one-way analysis of variance and the Tukey test.